Generation of single-cysteine E. coli ProQ variants to study RNA-protein interaction mechanisms

ProQ is a FinO-domain protein found in E. coli and other proteobacteria that has a global RNA-binding profile. In order to probe the detailed mechanism of RNA interactions, we have developed a collection of 13 E. coli ProQ variants that possess single-cysteine residues at varied positions on the surface of the N-terminal FinO domain and retain the ability to bind well to RNA. This set of variant ProQ proteins will support future biochemical and biophysical studies to map the orientation of bound RNAs to different sites around the ProQ protein, shedding light on the mechanism of ProQ-RNA interactions.

system to detect interaction between ProQ NTD and malM 3ʹUTR RNA, fused to the NTD of the α subunit of RNAP and an MS2 RNA hairpin (MS2 hp ; see methods), respectively.d-f) Results of β-galactosidase assays testing the interaction between ProQ NTD and malM-3'UTR using the B3H assay (Stockert et al. 2022;Pandey et al. 2020).The interactions of ProQ NTD mutants lacking one or both native cysteine residues are compared to wild-type (WT) ProQ.Data are plotted with varying levels of normalization: d) raw β-galactosidase data are shown for each transformation of KB473 reporter cells with pPrey-ProQ NTD (pKB951 or a mutant derivative) and pBait-malM-3' (pKB1210) or the corresponding negative controls (alpha empty and pCH1, respectively).The fold-stimulation over basal levels of lacZ activity is defined as the ratio of lacZ activity when both bait and prey are present divided by the highest value from the negative controls when either bait or prey is missing.e) B3H data of additional single-cysteine ProQ NTD mutants are plotted as fold-stimulation over basal levels of lacZ activity.f) B3H data of single and double-cysteine ProQ NTD mutants are plotted as normalized fold-stimulation values, where the fold-stimulation of ProQ WT is set to 1.0 and a fold-stimulation of 1.0, representing no B3H interaction, is set to 0 (see Methods).g) B3H data comparing interaction of ProQ C24S-C88A to versions of this cysteine-free ProQ NTD containing additional mutations introducing single cysteine residues.Data are plotted as normalized fold-stimulation values, where the fold-stimulation of ProQ C24S-C88A is set to 1.0.Data from mutants producing at least 70% of the interaction of ProQ C24S- C88A are shown in magenta and those <70% are shown in yellow.In c-f), bar graphs show the average and standard deviations of values collected from three or more independent experiments conducted across multiple days.h) Locations of residues substituted with single-cysteine mutations are shown on the AlphaFold-predicted structure (Jumper et al. 2021;Varadi et al. 2022); residues are colored as in (g) based on the strength of RNA-binding of the ProQ NTD variant with a cysteine at each position.

Description
RNA-binding proteins (RBPs) play essential roles in gene regulation in all organisms, including bacteria.Hfq is the most well characterized bacterial RNA-chaperone protein that stabilizes small RNAs (sRNAs) and facilitates sRNA base pairing with 5' untranslated regions (UTRs) of mRNAs.One class of bacterial RBPs that has garnered recent attention is FinO-domain proteins (Liao and Smirnov 2023); the E. coli ProQ protein is a global RBP with many sRNA and mRNA targets (Olejniczak and Storz 2017;Holmqvist et al. 2020) (Fig 1a,b).The core RNA-binding site of ProQ is found in a conserved concave pocket in the FinO-like N-terminal domain (NTD), which recognizes the 3' base and poly(U) tail of intrinsic terminators (Kim et al. 2022;Stein et al. 2023;Pandey et al. 2020).Still, several important questions remain unanswered about how ProQ interacts with RNA, such as: what role the C-terminal domain (CTD) and/or unstructured linker of E. coli ProQ play in RNA interactions; the degree of conformational flexibility and heterogeneity of ProQ-RNA interactions; whether RNA upstream of the intrinsic terminator participates in transient protein interactions; which surfaces ProQ uses to interact with RNA ligands that do not possess a 3' intrinsic terminator, such as fragments of 5' UTRs or open-reading frames (ORFs) (Holmqvist et al. 2018;Melamed et al. 2020); and whether ProQ can interact with two RNAs simultaneously or otherwise promote restructuring of RNAs.These mechanistic questions are important to fully understanding the role ProQ plays in regulating RNA stability and gene expression.
Several biochemical and biophysical techniques make use of site-directed bioconjugation of macromolecules for functionalization with small-molecule ligands (Hermanson 2013).For instance, site-specific hydroxyl radical probing with an FeBABE reagent can reveal details of RNA-protein interaction mechanisms (Duval et al. 2017).This approach makes use of the uniquely nucleophilic thiol group of cysteine side-chains (Gunnoo and Madder 2016): single-cysteine variants of a protein of interest are achieved by the removal of surface-exposed native cysteines and the installation of new cysteine residues that can be site-specifically labeled with a thiol-reactive FeBABE molecule (Duval et al. 2017).An important step in this process is identifying locations of cysteine variants that do not interfere with the native protein's function.In order to identify singlecysteine variants of E. coli ProQ that retain the ability to bind to RNA targets, we utilized a bacterial three-hybrid assay (B3H; (Berry and Hochschild 2018;Stockert et al. 2022) to test the RNA-binding activity of a panel of cysteine-free and singlecysteine variants of this protein.Briefly, this genetic assay makes use of a lacZ reporter gene where transcription is increased by interaction between a 'bait' RNA moiety and a 'prey' protein of interest (Fig 1c; see methods).
E. coli ProQ naturally possesses two cysteine residues-both in the N-terminal FinO domain (Fig 1a,b).We began by mutating each of these cysteine codons to either hydrophilic or hydrophobic amino acids (e.g.serine, leucine, valine or alanine) and testing their interactions in a B3H assay with the malM 3' UTR (Fig 1d,e).This RNA has been previously identified as a ProQ interactor in CLIP-seq and RIL-seq experiments (Holmqvist et al. 2018;Melamed et al. 2020) and its interaction with ProQ has been previously characterized in the B3H assay (Stein et al. 2020(Stein et al. , 2023)).In the B3H analysis of ProQ binding to malM-3', Cys24 was most robustly replaced by a serine residue (C24S), while Cys88 was best replaced by an alanine (C88A).A cysteine-free ProQ containing both of these substitutions together (C24S-C88A) retained ~70% of WT ProQ's interaction with malM-3' (Fig 1f).Using this cysteine-free C24S-C88A variant, we selected 14 additional residues that were predicted to be surface-exposed and not highly conserved to mutate to cysteine.These single-cysteine variants were tested for malM-3' interaction (Fig 1g).While some cysteine mutants were strongly disruptive to RNA interaction (e.g.G48C, D44C, S66C), 11 mutants retained at least 70% of the cysteine-free variant's interaction and were deemed suitable for future purification and site-directed conjugation, along with the C88A and C24S single-cysteine proteins.Importantly, this collection of 13 singlecysteine mutants encircles both the concave and convex faces of ProQ (Fig 1h).
Because the interaction of 3' UTRs with ProQ does not depend strongly on the CTD or unstructured linker (Pandey et al. 2020;Stein et al. 2020), we were unable to test single-cysteine mutants in these regions.However, it should be possible to introduce cysteine residues into positions of limited conservation that are predicted to be surface-exposed.One caveat to keep in mind is that ProQ variants may be less stable in vitro than they are when expressed inside of the cell as fusion proteins with α-NTD.It is also possible that a cysteine would be tolerated at a given position but that a larger moiety such as FeBABE or a fluorophore would sterically disrupt interaction.The RNA binding of site-specifically labeled proteins should therefore be confirmed under the conditions used for downstream biochemical or biophysical experiments.

Methods
Bacterial Strains and Plasmids.Plasmids, oligonucleotides and bacterial strains used in this study are provided in the Reagents section below.Site-directed mutants were constructed using Q5 site-directed mutagenesis (New England Biolabs) as previously described (Stein et al. 2023) with the specified mutagenic end-to-end primers, designed with NEBaseChanger.
β-galactosidase assays.RNA binding of ProQ mutants were assessed using a previously established bacterial three-hybrid (B3H) assay (Fig 1c) (Stockert et al. 2022;Berry and Hochschild 2018).Bait RNA is anchored to the B3H system by a DNA-RNA 'adapter' protein that consists of a fusion between the CI protein from bacteriophage  and the bacteriophage MS2 coat protein.Bait RNA is fused to an MS2 RNA hairpin (MS2 hp ).The prey protein is fused to the NTD of the RNA polymerase (RNAP) alpha subunit (α).Interaction between the bait RNA and prey protein is measured by the fold-stimulation of lacZ activity when both bait and prey are present in reporter cells over basal levels, defined by the highest value of negative controls when either bait or prey protein is missing.
For B3H assays, Δhfq plac-O L 2-62 reporter strain cells (KB473) were transformed with three compatible plasmids: first by pAdapter (pCW17), and subsequently with a combination pPrey and pBait plasmids.pPrey plasmids encoded either the WT α-ProQ NTD fusion protein (resi=2-131; pKB951 or a site-specific mutant derivative) or α alone as a negative control.pBait plasmids encoded a hybrid RNA with the 3' UTR of malM following the MS2 hp (pKB1210) or an RNA that contained only the MS2 hp moiety (pCH1) as a negative control.Transformations were conducted in 96-well plates as previously described (Stockert et al. 2022).Overnight cultures were directly inoculated with transformants and grown in 1 mL of LB supplemented with carbenicillin (100 μg/mL), chloramphenicol (25 μg/mL), kanamycin (50 μg/mL), spectinomycin (100 μg/mL) and 0.2% arabinose.Plates were sealed with breathable film and grown ON at 37℃, shaking at 900 rpm.In the morning, day cultures (200 μL) were inoculated with 10 μL of ON culture, covered with a plastic lid and grown at 37℃, shaking at 900 rpm until OD 600 values reached mid-log (0.3-0.6).Cells were lysed with the addition of 10 μL of PopCulture Reagent (Novagen) and of 4 units of rLysozyme (Novagen).β-gal activity was measured as previously described (Stockert et al. 2022).
The β-gal activity produced by reporter cells transformed with each combination of bait RNA and prey protein was divided by the highest value of the two corresponding negative controls to yield fold-stimulation over basal lacZ levels.These foldstimulation values were then normalized using the equation: (fold-stimulation mutant -1)/(fold-stimulation comparison -1), where the point of comparison was either the fold-stimulation produced by ProQ WT or ProQ C24S-C88A .This normalization sets the B3H value for the highest value to 1.0 and the lack of a detectable interaction (1-fold-stimulation of lacZ) equal to 0.
Protein Structures.The positions of native and introduced cysteine residues were mapped onto the AlphaFold predicted structure of E. coli ProQ (Jumper et al. 2021;Varadi et al. 2022), which has been found to be consistent with genetic mutagenesis data (Stein et al. 2023).Structures were rendered in the PyMOL Molecular Graphics System (Schrödinger, LLC).

Figure 1 .
Figure 1.Testing the RNA-binding ability of E. coli ProQ single-cysteine variants: a) Domain-and b) protein-structure of E. coli ProQ, with the position of the two native cysteine residues highlighted.The 3D protein structure was predicted by AlphaFold (Jumper et al. 2021; Varadi et al. 2022).c) Schematic of bacterial three-hybrid

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Encodes full-length lCI under the control of the lacUV5 promoter; confers CamR (Dove et al. 1997) pBrα Empty vector Encodes full-length rpoA under the control of tandem lpp and lacUV5 promoters248 of CI fused to an MS2 coat protein; transcription of this protein under the control of a constitutive promoter; confers CmR (Pandey et al., 2020) pKB951 pBrα-ProQ NTD Encodes residues 1-248 of alpha fused to residues 1-131 of wild type E. coli proQ; TAA stop codon after proQ added through PCR; of E. coli malM (final 90 nts) cloned behind MS2hp in pCH1 between XmaI/ HindIII sites; RNA encodes its own terminator; confers SpcR (Stein et al. 2020) pASW24 pBrα-ProQ NTD -C24S proQ C24S mutation introduced to